D-ketohexose 3-epimerase, and its preparation

ABSTRACT

D-ketohexose 3-epimerase, a novel epimerase, is obtained by cultivating bacteria of the genus Pseudomonas including Pseudomonas cichorii ST-24 (FERM BP-2736). D-Ketohexose 3-epimerase epimerizes D-ketohexose, D-ketopentose and L-ketopentose at their C-3 positions to form their corresponding epimeric counterparts in a high yield at a high conversion rate. Interconversion reaction using D-Ketohexose 3-epimerase yields mixture of intact ketose and its epimeric counterpart which can impart an appropriate sweetness, gloss and improve taste quality when used in foods, beverages, feeds, pet foods, dentifrice, cachou, sublingual agents and internal medicines.

BACKGROUND OF THE INVENTION

1. Field of the invention

The present invention relates to D-ketohexose 3-epimerase, and itspreparation and uses.

2. Description of the prior art

As described in Enzyme Nomenclature published by Academic Press Inc.,USA, 1992, epimerases act on various saccharides. Since conventionalepimerases such as ribulose-phosphate 3-epimerase (EC 5.1.3.1) and UDPglucose 4-epimerase (EC 5.1.3.2), however, mainly act on saccharidescoupled with phosphates or UDP, they are not usable in industrial-scaleproduction of free neutral saccharides. There has been known only twoepimerases, i.e., aldose 1-epimerase (EC 5.1.3.3) and cellobioseepimerase (EC 5.1.3.11), which act on free saccharides. The formercatalyzes the epimerization between α- and β-anomers of aldoses at theirC-1 positions, while the latter catalyzes the epimerization between α-and β-anomers of cellobiose. Although there has been a great demand forepimerases which act on free ketoses, the existence of such has not beenconfirmed.

It has been in a great demand to obtain a ketose epimerase which acts onfree ketoses, and also to establish its preparation and uses.

SUMMARY OF THE INVENTION

We have screened various epimerases which readily epimerize freeketopentoses and ketohexoses into their corresponding epimericketopentoses and ketohexoses. As a result, we eventually foundD-ketohexose 3-epimerase and established its production as well asestablishing a method of converting D-ketohexose, D-ketopentose andL-ketopentose using the enzyme, a process of producing convertedketoses, and a process of producing sweetener containing the same. Thus,we completed this invention. More particularly, the D-ketohexose3-epimerase of the invention is a novel enzyme which has an activity ofepimerizing D-ketohexose at its C-3 position into its correspondingepimeric D-ketohexose, and shows the following physicochemicalproperties:

1. Action and substrate specificity

Epimerizing D-ketohexose at its C-3 position into its correspondingepimeric D-ketohexose.

Epimerizing D- and L-ketopentoses at their C-3 positions into theircorresponding epimeric D- and L-ketopentoses;

2. Optimum pH and pH stability

Possessing an optimum pH of 7-10 and being stable at pH 5-10;

3. Optimum temperature and thermal stability

Possessing an optimum temperature of around 60° C., and being stable attemperature up to 50° C.; and

4. Ultraviolet absorption spectrum

Exhibiting an absorption at a wavelength of 275-280 nm.

BRIEF EXPLANATION OF THE ACCOMPANYING DRAWINGS

FIG. 1 shows the optimum pH of the present enzyme.

FIG. 2 shows the pH stability of the present enzyme.

FIG. 3 shows the optimum temperature of the present enzyme.

FIG. 4 shows the thermal stability of the present enzyme.

FIG. 5 shows the molecular weight of the present enzyme.

FIG. 6 shows a polyacrylamide gel electrophoretic pattern of the presentenzyme.

FIG. 7 shows an infrared absorption spectrum of D-sorbose prepared fromD-tagatose by the invention.

FIG. 8 shows an infrared absorption spectrum of a standard D-sorbose.

In FIG. 5, the symbol "A" shows bovine serum albumin (BSA); the symbol"B", ovalbumin; the symbol "C", the present enzyme (D-ketohexose3-epimerase); the symbol "D", chymotrypsinogen A; and the symbol "E",cytochrome C.

In FIG. 6, the symbol "I" shows the starting point of electrophoresis;the symbol "II", the present enzyme (D-ketohexose 3-epimerase); and thesymbol "III", bromophenol blue.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to D-ketohexose 3-epimerase, and itspreparation and uses.

We have screened various epimerases which readily epimerize freeketopentoses and ketohexoses into their corresponding epimericketopentoses and ketohexoses. As a result, we eventually foundD-ketohexose 3-epimerase and established its production as well asestablishing a method of converting D-ketohexose, D-ketopentose andL-ketopentose using the enzyme, a process of producing convertedketoses, and a process of producing sweetener containing the same. Thus,we completed this invention. More particularly, the D-ketohexose3-epimerase of the invention is a novel enzyme which has an activity ofepimerizing D-ketohexose at its C-3 position into its correspondingepimeric D-ketohexose, and shows the following physicochemicalproperties:

1. Action and substrate specificity

Epimerizing D-ketohexose at its C-3 position into its correspondingepimeric D-ketohexose.

Epimerizing D- and L-ketopentoses at their C-3 positions into theircorresponding epimeric D- and L-ketopentoses;

2. Optimum pH and pH stability

Possessing an optimum pH of 7-10 and being stable at pH 5-10;

3. Optimum temperature and thermal stability

Possessing an optimum temperature of around 60° C., and being stable attemperature up to 50° C.; and

4. Ultraviolet absorption spectrum

Exhibiting an absorption at a wavelength of 275-280 nm.

The D-ketohexose 3-epimerase according to the present invention isusually obtained by the cultivation of microorganisms capable ofproducing D-ketohexose 3-epimerase.

Examples of microorganisms advantageously usable in the invention arebacteria of the genus Pseudomonas including Pseudomonas cichorii ST-24(FERM BP-2736) and its mutants disclosed in Japanease Patent Laid-OpenNo.266,996/91.

D-ketohexose 3-epimerase is prepared by cultivating such a bacterium ina nutrient culture medium containing carbon sources, nitrogen sources,minerals, vitamins and the like for about 1-5 days, preferably, underaerobic conditions such as aeration-agitation conditions; and recoveredfrom obtained cells and/or supernatant. The resultant culture is usableas a crude D-ketohexose 3-epimerase. If necessary, the culture can bepartially purified by conventional methods such as filtration,centrifugation, salting out, dialysis, concentration and lyophilization,prior to its use. In case of a higher purification is required, theculture is purified to the possible highest level by absorption anddesorption using an ion-exchanger, gel filtration, isoelectricfocussing, electrophoresis, high-performance liquid chromatography(hereinafter abbreviated as "HPLC"), affinity chromatography, and/orabsorption and desorption using monoclonal antibodies.

In the conversion reaction wherein one or more members selected from thegroup consisting of D-ketohexose, D-ketopentose and L-ketopentose areepimerized at their C-3 positions into their corresponding epimericD-ketohexose, D-ketopentose and L-ketopentose; D-ketohexose 3-epimerasecan immobilized in usual manner and advantageously used in repeated orcontinuous reaction.

The present conversion reaction is usually carried out under thefollowing conditions: Substrate concentration is set in the range of1-60 w/v %, preferably, about 5-50 w/v %; reaction temperature, in therange of 10°-70° C., preferably, about 30°-60° C.; reaction pH, in therange of 5-10, preferably, in the range of about 7-10; and the amount ofenzyme, at least one unit per gram of substrate, preferably, 50-5,000units per gram of substrate. The reaction time can be arbitrarilychosen, usually, in the range of 5-50 hours in a batch reaction from aneconomical view-point.

Reaction mixtures obtainable by the present conversion reaction, whichcontain newly formed ketoses and intact ketoses as a starting material,can be advantageously used intact as a sweetener, moisture-impartingagent, crystallization-preventing agent and gloss-imparting agent. Thereaction mixtures are usually prepared in usual manner into syrupyproducts by successive decoloration with activated carbons, salting outwith ion-exchange in H- and OH-form, and concentration.

If necessary, the concentrates thus obtained can be easily or readilyseparated and purified on a column chromatography using strongly-acidiccation exchange of alkaline metal- or alkaline earth metal-form toobtain the newly formed ketoses and the intact ketoses as a startingmaterial, followed by concentrating the ketose-rich fractions intosyrupy products. If ketoses are crystallizable, they are advantageouslycrystallized into crystalline products. Separated ketoses can beadvantageously used as a starting material for the next conversionreaction.

The separated ketoses are advantageously usable as a sweetener, inparticular, to impart an appropriate sweetness to orally administrableproducts such as foods, beverages, feeds, pet foods, dentifrice, cachou,sublingual agents and internal medicines, as well as to improve theirtaste qualities. The ketoses can be also advantageously used as a carbonsource for fermentation, as well as chemical reagent, material andintermediate for chemicals and pharmaceuticals.

The following examples will explain the present invention.

EXAMPLE 1

A nutrient culture medium consisting of 0.2 w/v % ammonium sulfate, 0.24w/v % potassium phosphate monobasic, 0.56 w/v % potassium phosphatedibasic, 0.01 w/v % magnesium sulfate heptahydrate, 0.5 w/v % yeastextract, 1 w/v % D-glucose and deionized water was placed in a jarfermenter, sterilized at 120° C. for 20 minutes, and asepticallyinoculated with 1 v/v % of a seed culture of Pseudomonas cichorii ST-24(deposited with Fermentation Research Institute, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry,1-3, Higashi, 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan on Jan. 19,1990 and given the accession number FERM BP-2736), followed by thecultivation at 30° C. for 40 hours under aeration-agitation conditions.The cells which had been recovered from 80 liters of the resultantculture were crushed by grinding in the presence of activated aluminaand then subjected to extract an enzyme in 50 mM Tris-HCl buffer (pH7.5).

The obtained crude enzyme solution was purified in the presence ofmanganese chloride by repeated fractional sedimentation usingpolyethylene glycol 6,000 (hereinafter abbreviated as "PEG"). Theprecipitates, which had formed in the crude enzyme solution at the PEGconcentrations of 5-18 w/v % in the presence of 0.1M manganese chloride,were dissolved in a fresh preparation of the same buffer. The crudeenzyme solution was further purified by the above fractionalsedimentation. The resultant solution was heated at 50° C. for 20minutes, and the resultant degenerated proteins were removed bycentrifugation. The resultant product was purified by allowing it toabsorb on "DEAE-TOYOPEARL® 650M", a product of Tosho Corporation, Tokyo,Japan, and eluting the absorbed substance with potassium chloridesolution. The purified product thus obtained was demineralized on anultrafiltration using "Toyo Roshi UK-10", a filter membranecommercialized by Toyo Roshi Kaisha Ltd., Tokyo, Japan, concentrated andpurified on a gel filtration using "Sephadex® G150", a product ofPharmacia, Uppsala, Sweden.. Fractions with an activity wereconcentrated and purified on an isoelectric focussing using"Ampholine®", a product of Pharmacia, Uppsala, Sweden.

The enzyme specimen thus obtained was assayed as follows: The reactionsolution used in this assay consisted of 100 microliters 50 mM Tris-HClbuffer (pH 7.5), 50 microliters 40 mM D-tagatose and 50 microliters ofan enzyme solution. The enzymatic reaction was carried out at 30° C. for60 minutes, and the formed D-sorbose was quantitated on HPLC. One unitof the enzyme activity is defined as the amount of enzyme thatepimerizes one micromol of D-tagatose per one minute.

The purification procedure was as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                            Enzyme           Purification                             Purification                                                                              Protein activity   Yield degree                                   step        (mg)    (× 10.sup.3 -unit)                                                                 (%)   (fold)                                   ______________________________________                                        Crude extract                                                                             42,100  52,000     100.0 1                                        PEG (first) 8,380   50,600     97.3  4.9                                      PEG (second)                                                                              3,760   26,400     50.8  5.7                                      Heat treatment                                                                            3,210   27,800     53.5  7.0                                      DEAE-         415   17,200     33.1  33.6                                     TOYOPEARL ®                                                               Sephadex ® G150                                                                         127   16,800     32.3  106                                      Isoelectric focussing                                                                      3.0     1,078     2.1   290                                      ______________________________________                                    

As obvious from the results in Table 1, the purification step improvedthe specific activity of a crude enzyme by about 290 folds, and gave theyield of about 2%. The physicochemical properties of the presentD-ketohexose 3-epimerase were studied with the finally obtained sampleof these purification steps:

(1) Action and substrate specificity

D-ketohexose 3-epimerase acts on all of the four D-ketohexoses(D-tagatose, D-sorbose, D-fructose and D-psicose) among eight D- andL-ketohexoses, and epimerizes them at their C-3 positions into theircorresponding epimeric D-ketohexoses. It acts on all four D- andL-ketopentoses (D- and L-xyluloses, D- and L-ribuloses), and epimerizesthem at their C-3 positions into their corresponding epimeric D- andL-ketopentoses.

The enzyme most strongly catalyzed each interconversion reaction ofD-tagatose and D-sorbose. The activity in the interconversion reactionsof D-fructose and D-psicose was about 30-40% of that in eachinterconversion reaction D-tagatose and D-sorbose. While the activity inthe interconversion reaction between D- and L-ketohexoses was about10-30% of that in D-tagatose and D-sorbose. All the reactions werereversible- and equilibrium-reactions and did not require coenzymes andmetal ions.

(2) Optimum pH and pH stability

The Optimum pH of the enzyme was determined in accordance with theaforementioned enzyme assay. The results were as shown in FIG. 1. In thefigure, the symbol "-□-" shows 50 mM citrate buffer; the symbol "- -",50 mM malonate buffer; the symbol "-◯-", 50 mM Tris-HCl buffer; and thesymbol "- -", 50 mM glycine-NaOH buffer. As is obvious from FIG. 1, theoptimum pH was 7-10.

The pH stability of the enzyme was studied by incubating it at 30° C.for 60 minutes, then measuring the residual activity. The results wereas shown in FIG. 2. As is obvious from FIG. 2, the enzyme was stable atpH 5-10.

(3) Optimum temperature and thermal stability

The optimum temperature of the enzyme was determined in accordance withthe aforementioned enzyme assay. From the results as shown in FIG. 3,the optimum temperature was around 60° C. The thermal stability wasstudied by incubating it in 50 mM Tris-HCl buffer (pH 7.5) at differenttemperatures for 10 minutes, then determining the residual activity. Asis obvious from FIG. 4, the enzyme was stable at a temperature up to 50°C.

(4) Ultraviolet absorption spectrum

The enzyme exhibits an absorption at a wavelength of 275-280 nm.

(5) Molecular weight

The molecular weight of the enzyme was determined on a gel filtrationchromatography using a column (1.2×100 cm) packed with "Sephadex® G150"wherein 25 mM Tris-HCl buffer (pH 7.5) was used as an eluent andsubjected to the column at a flow rate of 0.15 ml/min. The results wereas shown in FIG. 5. In the figure, the symbol "A" shows bovine serumalbumin, 67,000 dalton; the symbol "B", ovalbumin, 45,000 dalton; thesymbol "C", the present enzyme; the symbol "D", chymotrypsinogen A,25,000 dalton; and the symbol "E", cytochrome C, 12,500 dalton. As isobvious from FIG. 5, the molecular weight of the enzyme (C) wasdetermined as 41,000±3,000, based on a standard curve which was made byplotting the molecular weights of proteins A, B, D and E which had beenknown.

(6) Isoelectric point

Upon analysis on an isoelectric focussing using "Ampholine®", the enzymeshowed an isoelectric point of the enzyme is pH 4.3±0.2.

(7) Polyacrylamide gel electrophoresis

FIG. 6 shows a polyacrylamide gel electrophoretic pattern of the enzyme.In the figure, the symbol "I" shows the starting point; the symbol "II",the present enzyme; and the symbol "III", bromophenol blue. As isobvious from FIG. 6, the enzyme showed a single band.

EXAMPLE 2 Interconversion reaction of D-tagatose and D-sorbose

Five w/v % aqueous D-tagatose solution (pH 7.5) was added with 500units/g D-tagatose of a partially purified enzyme solution which hadbeen subjected to the purification steps up to the second fractionationusing PEG in Example 1, and the mixture was enzymatically reacted at 40°C. for 30 hours. After completion of the reaction, the resultantreaction mixture was decolored with activated carbons in usual manner,demineralized with "Diaion SK1B (H-form)" and "Diaion WA30 (OH-form)",products of Mitsubishi Chemical Industries Ltd., Tokyo, Japan, andconcentrated in vacuo to obtain about 60%, transparent syrup containingD-tagatose and D-sorbose. The syrup was separated and purified on acolumn chromatography using "Dowex 50W-X4", a strongly-acidic cationexchange of Dow Chemical Company, Midland, Mich., USA, concentrated,crystallized and separated to obtain a crystalline D-sorbose in theyield of about 55%. The physicochemical properties of the product wereas follows: The melting point was 165° C. and the specific rotation was[α]_(D) ²⁰ =+44.0° (C=10% H₂ O) The infrared absorption spectrum (thedatum of the product was shown in FIG. 7, and D-sorbose as a standardreagent was shown in FIG. 8.) had a good agreement with that ofD-sorbose. Based on these results, the resultant saccharide convertedfrom D-tagatose by the enzyme was D-sorbose. The product is favorablyused as a sweetener, carbon source for fermentation, chemical regent,material and intermediate in chemicals and pharmaceuticals. The enzymereaction is a reversible reaction, and because of this, D-tagatose isreadily obtained when D-sorbose is used as a starting material.

EXAMPLE 3 Interconversion reaction of D-fructose and D-psicose

Ten w/v % aqueous D-fructose solution (pH 7.5) was added with 1,500units/g D-fructose a partially purified enzyme solution which had beensubjected to the purification steps up to the purification usingDEAE-TOYOPEARL® in Example 1, and the mixture was enzymatically reactedat 45° C. for 30 hours. After completion of the reaction, the resultantreaction mixture was similarly as in Example 2, decolored, demineralizedand concentrated in vacuo to obtain a transparent syrup containingD-psicose. The syrup was separated and purified on a columnchromatography using a strongly-acidic cation exchange similarly as inExample 2 to obtain a syrupy D-psicose in the yield of about 25%, on adry solid basis (d.s.b.). The physicochemical properties had a goodagreement with those of the standard D-psicose. The product is favorablyused as a sweetener, carbon source for fermentation, chemical regent,material and intermediate for chemicals and pharmaceuticals. The enzymereaction is a reversible reaction, and because of this, D-fructose isreadily obtained when D-psicose is used as a starting material.

EXAMPLE 4 Interconversion reaction of D-fructose and D-psicose

Ten w/v % aqueous D-fructose solution (pH 7.0) was added with 1,000units/g D-fructose of a partially purified enzyme solution which hadbeen subjected to the purification steps up to the second fractionationusing PEG in Example 1, and the mixture was enzymatically reacted at 50°C. for 30 hours. After completion of the reaction, the resultantreaction mixture was similarly as in Example 2, decolored, demineralizedand concentrated in vacuo to obtain a transparent syrup containingD-fructose and D-psicose in the yield of about 90%, d.s.b. The productis suitably used as a high-quality sweetener with a highsweetening-power, and advantageously used as a moisture-imparting agent,a crystallization-preventing agent and a gloss-imparting agent, as wellas a sweetening agent for foods and beverages.

EXAMPLE 5 Interconversion reaction of D-xylulose and D-ribulose

One w/v % aqueous D-xylulose solution (pH 7.5) was added with 3,000units/g D-xylulose of a partially purified enzyme solution which hadbeen subjected to the purification steps up to the purification usingDEAE-TOYOPEARL® in Example 1, and the mixture was enzymatically reactedat 35° C. for 50 hours. After completion of the reaction, the resultantreaction mixture was similarly as in Example 2, decolored, demineralizedand concentrated in vacuo to obtain a transparent syrup containingD-ribulose. Similarly in Example 2, the syrup was separated and purifiedon a column chromatography using a strongly-acidic cation exchange toobtain a syrupy D-ribulose in the yield of about 20%, d.s.b. Thephysicochemical properties had a good agreement with those of thestandard D-ribulose. The product is favorably used as a sweetener,carbon source for fermentation, regent, material and intermediate forchemicals and pharmaceuticals. The enzyme reaction is a reversiblereaction, and because of this, D-xylulose is readily obtained whenD-ribulose is used as a starting material.

EXAMPLE 6 Interconversion reaction of L-xylulose and L-ribulose

One w/v % aqueous L-xylulose solution (pH 7.5) was added with 3,000units/g L-xylulose of a partially purified enzyme solution which hadbeen subjected to the purification steps up to the purification usingDEAE-TOYOPEARL® in Example 1, and the mixture was enzymatically reactedat 35° C. for 50 hours. After completion of the reaction, the resultantreaction mixture was similarly as in Example 2, decolored, demineralizedand concentrated in vacuo to obtain a transparent syrup containingL-ribulose. Similarly as in Example 2, the syrup was separated andpurified on a column chromatography using a strongly-acidic cationexchange to obtain a syrupy L-ribulose in the yield of about 20%, d.s.b.The physicochemical properties have a good agreement with those of thestandard L-ribulose. The product is favorably used as a sweetener,carbon source for fermentation, chemical regent, material andintermediate for chemicals and pharmaceuticals. The enzyme reaction is areversible reaction, and because of this, L-xylulose is readily obtainedwhen L-ribulose is used as a starting material.

When the D-ketohexose 3-epimerase according to the present invention isallowed to act on free D-ketohexose, D-ketopentose and L-ketopentose,these ketoses are epimerized at their C-3 positions to readily formtheir corresponding epimeric D-ketohexose, D-ketopentose andL-ketopentose. The reaction system would open the way to anindustrial-scale production of ketoses which has been deemed verydifficult. Thus the finding of D-ketohexose 3-epimerase, andestablishment of the preparation and uses have a great significance inthe fields of saccharide-manufacturing industries, as well as food-,pharmaceutical- and cosmetic-industries.

While there has been described what is at present considered to be thepreferred embodiments of the invention, it will be understood thevarious modifications may be made therein, and it is intended to coverin the appended claims all such modifications as fall within the truespirits and scope of the invention.

We claim:
 1. A purified D-ketohexose 3-epimerase having the following physicochemical properties:(a) ActivityEpimerizing free D-ketohexose at its C-3 position into its corresponding epimeric D-ketohexose; and epimerizing free D- and L- ketopentoses at their C-3 positions into their corresponding epimeric D- and L- ketopentoses; (b) Optimum pHA pH of 7-10; (c) pH stabilityStable at a pH of 5-10; (d) Optimum temperaturesAround 60° C.; (e) Thermal stabilityStable at a temperature of 50° C.; (f) Ultraviolet absorption spectrumExhibiting an absorption peak at a wavelength of 275-280 nm; (g) Molecular weight
 41. 000±3,000 daltons on gel filtration chromatography; and(h) Isoelectric point4.3±0.2.
 2. A purified D-ketohexose 3-epimerase in accordance with claim 1 obtainable from Pseudomonas cichorii ST-24, FERM BP-2736.
 3. A process to prepare D-ketohexose 3-epimerase, which comprises:(a) cultivating in a nutrient culture medium a bacterium which is Pseudomonas cichorii St-24 (FERM BP-2736) capable of producing D-ketohexose 3-epimerase to form D-ketohexose 3-epimerase; and (b) recovering the resultant D-ketohexose 3-epimerase having all of the properties as recited in claim
 1. 